Supplementary Materials for

Transcription

1 Supplementary Materials for STAT3 Induction of mir-146b Forms a Feedback Loop to Inhibit the NF-κB to IL-6 Signaling Axis and STAT3-Driven Cancer Phenotypes Michael Xiang, Nicolai J. Birkbak, Vida Vafaizadeh, Sarah R. Walker, Jennifer E. Yeh, Suhu Liu, Yasmin Kroll, Mark Boldin, Konstantin Taganov, Bernd Groner, Andrea L. Richardson, David A. Frank* *Corresponding author. david_frank@dfci.harvard.edu Published 28 January 2014, Sci. Signal. 7, ra11 (2014) DOI: /scisignal Thes PDF file includes: Fig. S1. Doxycycline-inducible cell systems for mirna screening and analyses of mir-146b regulation. Fig. S2. STAT target gene expression across mouse mammary developmental stages. Fig. S3. Western blotting of cells after cytokine stimulation. Fig. S4. Characterization of mir-146b promoter methylation. Fig. S5. Effects of mir-146b mimic and IL-6 neutralizing antibody on STAT3 phosphorylation and gene expression. Fig. S6. Effect of mir-146b mimic and JAK inhibitor in SUM-159 cells. Fig. S7. Analysis of triple-negative tumors versus other molecular subtypes in TCGA breast cancer cohort. Table S1. Results of mirna screen. Table S2. Primers for qrt-pcr.

2 Supplementary Materials Fig. S1. Doxycycline-inducible cell systems for mirna screening and analyses of mir-146b regulation. (A) Induction of FLAG-tagged STAT3C in doxycycline (Dox)-inducible cells after 24 hours. (B) Induction of SOCS3, a STAT3 target gene, in doxycycline-inducible cells after treatment with doxycycline (2 µg/ml) for hours. (C) Screening strategy to identify STAT3- regulated mirnas. (D) Analysis of direct and indirect STAT targets using the protein synthesis inhibitor cycloheximide (CHX) in MCF-10A cells pre-treated with vehicle or 2 µg/ml CHX for 1 1

3 hour, then stimulated with 20 ng/ml IL-6 for 2 hours. Primary transcripts of validated mirna hits from the screen were analyzed by qrt-pcr. (E-F) MiR-146b expression in MCF10A cells transfected with Dox-inducible constitutively active STAT mutants (STAT3C and STAT5A1*6). (G) MiR-146a and mir-146b expression in immortalized mammary epithelial cells (IMECs) treated with TNF-α (10 ng/ml) for 48 hours. Data in (B) and (D) to (G) are means ± SE of one of two independent experiments. 2

4 Relative expression beta-casein Relative expression SOCS3 Fig. S2. STAT target gene expression across mouse mammary developmental stages. The gene CASB, which encodes beta-casein (left), is a STAT5 target, and SOCS3 (right) is a STAT3 and STAT1 target. Data are means ± SE of technical replicates from one mouse, representative of two mice. 3

5 Fig. S3. Western blotting of cells after cytokine stimulation. STAT activation was induced in cells that lack basal STAT activation after treatment with IL-6 or IFN-γ (20 ng/ml) for 15 min. Immunoblotting was performed to detect phospho-tyrosine (p) STAT3 or STAT1 against total (t) STAT3 or STAT1. Data are representative of 2 experiments. 4

6 Fig. S4. Characterization of mir-146b promoter methylation. (A) SssI-treated plasmid DNA was resistant to digestion by methylation-sensitive HpaII restriction enzyme, providing verification of methylation. HpaII is a 4-base cutter and therefore the average fragment size is predicted to be 256 bp. This is below the size of the smallest fragments retained on the gel (500 bp, as indicated). (B) Analysis of SOCS3 expression as in Fig. 3B. Data are means ± SE of one of two independent experiments. (C) Correlation between mir-146b expression and extent of CpG methylation at 22 CpGs within 10 kb of mir-146b interrogated by Illumina 450k profiling in TCGA. Sites highlighted in blue indicate a highly significant inverse correlation. (D) Lack of correlation between CpG methylation at the mir-146b locus and mir-21 abundance in TCGA. 5

7 (E) Strong positive correlation between CpG sites near mir-146b, indicating coordinate regulation in this region in TCGA. A MDA-468 BT-549. Mimic: ctrl 146b ctrl 146b pstat3 Y705 pstat3 S727 tstat3 TRAF6 IRAK1 actin C 2 Relative expression Relative expression B control mimic mir-146b mimic mir-146b mimic + TNF-α EGR1 STAT3 KLF4 SOCS3 BCL6 BCL3 BCL-X D IgG Anti-IL-6 pstat3 Y705 tstat3 IL6 control mimic #2 mir-146b mimic MDA-468 BT CCL2 IL6 SOCS3 JUNB Fig. S5. Effects of mir-146b mimic and IL-6 neutralizing antibody on STAT3 phosphorylation and gene expression. (A) An immunoblot of breast cancer cells (MDA-MB- 468 and BT-549) transfected with mir-146b or control mimic (25 nm) for 72 hours. (B) Expression of STAT3 target genes in MDA-MB-453 cells 72 hours after transfection with mir- 146b mimic. (C) Expression of indicated NF-κB and STAT3 target genes in SUM-159 cells 72 hours after transfection with mirna mimic alone or with concurrent TNF-α treatment. Data are means ± SE of one of 2 experiments. (D) An immunoblot of breast cancer cells (MDA-MB-468 6

8 and BT-549) treated with 3 µg/ml IL-6 neutralizing antibody or control IgG for 6 hours in fresh media. Data are representative of at least two independent experiments. 7

9 A B Relative viability ctrl 146b ctrl 146b Relative expression vehicle JAK inhibitor EV cells STAT3C cells Fig. S6. Effect of mir-146b mimic and JAK inhibitor in SUM-159 cells. (A) Cell viability (as a measure of ATP) in SUM-159 cells transfected with mir-146b or control mimic (25 nm) for 48 hours, when the cells were also assessed motility, and using Cell Titer Glo. Data are mean ± SD of 2 experiments. Data are mean ± SD of three biological replicates. (B) Expression of mature mir-146b and mir-146b primary transcript analyzed by qrt-pcr in SUM-159 cells treated with JAK inhibitor 1 (1 µm) for 72 hours. Data are means ± SEM of one representative biological replicate from two independent experiments. 8

10 A p = 0.60 p = 1.2x10-5 p = 2.9x10-7 B Gene rho p-value BIRC x10-25 RELB x10-16 IL x10-15 PTGS2 (COX2) x10-55 IL-6 expression (log2) TNFAIP3 (A20) x10-42 CCL x10-59 CCL x10-23 CCL x10-15 CCL x10-33 CCL x10-50 CXCL x10-43 CXCL x10-47 CCR x10-17 ER/PR+ HER2 Triple negative HAS x10-28 PIM x10-36 VIM (vimentin) x10-16 C mir-146b percentile: Fraction surviving p = 0.03 Triple negative tumors only Days Fig. S7. Analysis of triple-negative tumors versus other molecular subtypes in TCGA breast cancer cohort. (A) Analysis of IL6 expression performed as in Fig. 7B, but comparing triple negative breast cancers to other molecular subtypes. (B) Correlation of IL-6 abundance with expression of the indicated NF-κB target genes. (C) Survival analysis against mir-146b expression in patients as in Fig. 7C, but in the specific subset of triple negative breast cancers. 9

11 Table S1. Results of mirna screen. Shown are ratios for each detected mirna in doxycycline-induced MCF-10A cells compared to untreated cells. The housekeeping genes RNU44 and RNU48 were used for normalization. The raw data for the screen, which is deposited to GEO (GSE44089), consist of TaqMan qrt-pcr Ct values for each microrna assayed, or no value/ Undetermined if the microrna was not detected within 40 cycles of amplification. Each condition was performed as a biological triplicate. Ct values greater than 30 are considered unreliable based on the experience of the profiling core, due to the large within-sample variation at this expression level. MicroRNA Fold-change (average across all replicates) Error bar (SD) of untreated replicates Error bar (SD) of doxycycline replicates hsa-let-7a hsa-let-7b hsa-let-7c hsa-let-7d hsa-let-7e hsa-let-7f hsa-let-7g hsa-mir hsa-mir hsa-mir hsa-mir-106a hsa-mir-106b hsa-mir hsa-mir-10a hsa-mir-125a-3p hsa-mir-125a-5p hsa-mir-125b hsa-mir hsa-mir hsa-mir-130a hsa-mir-130b hsa-mir hsa-mir-135a hsa-mir-135b hsa-mir

12 hsa-mir hsa-mir-139-5p hsa-mir-140-3p hsa-mir-140-5p hsa-mir hsa-mir-142-3p hsa-mir hsa-mir-146a hsa-mir-146b-5p hsa-mir-148a hsa-mir-148b hsa-mir hsa-mir hsa-mir hsa-mir-15a hsa-mir-15b hsa-mir hsa-mir hsa-mir-181a hsa-mir-181c hsa-mir hsa-mir hsa-mir hsa-mir hsa-mir-18a hsa-mir-18b hsa-mir hsa-mir hsa-mir hsa-mir-193a-3p hsa-mir-193a-5p hsa-mir-193b hsa-mir hsa-mir hsa-mir-196b hsa-mir hsa-mir-19a hsa-mir-19b hsa-mir-200b hsa-mir-200c hsa-mir hsa-mir hsa-mir

13 hsa-mir-20a hsa-mir-20b hsa-mir hsa-mir hsa-mir hsa-mir hsa-mir hsa-mir p hsa-mir hsa-mir hsa-mir hsa-mir hsa-mir hsa-mir-23b hsa-mir hsa-mir hsa-mir-26a hsa-mir-26b hsa-mir-27a hsa-mir-27b hsa-mir-28-3p hsa-mir-28-5p hsa-mir-296-3p hsa-mir-296-5p hsa-mir-29a hsa-mir-29b hsa-mir-29c hsa-mir-301a hsa-mir-301b hsa-mir-30b hsa-mir-30c hsa-mir hsa-mir-320a hsa-mir-323-3p hsa-mir hsa-mir-324-3p hsa-mir-324-5p hsa-mir hsa-mir-330-3p hsa-mir-331-3p hsa-mir-331-5p hsa-mir hsa-mir-339-3p

14 hsa-mir-339-5p hsa-mir hsa-mir-342-3p hsa-mir hsa-mir-34a hsa-mir-34c-5p hsa-mir-361-5p hsa-mir-362-3p hsa-mir-362-5p hsa-mir hsa-mir-374a hsa-mir-374b hsa-mir hsa-mir-376a hsa-mir-376c hsa-mir hsa-mir hsa-mir hsa-mir-422a hsa-mir-423-5p hsa-mir hsa-mir hsa-mir-449a hsa-mir-449b hsa-mir-450a hsa-mir hsa-mir hsa-mir-455-3p hsa-mir-455-5p hsa-mir hsa-mir-485-3p hsa-mir-487b hsa-mir hsa-mir-491-5p hsa-mir hsa-mir hsa-mir hsa-mir-502-3p hsa-mir-502-5p hsa-mir hsa-mir hsa-mir hsa-mir-508-3p

15 hsa-mir-509-5p hsa-mir-517c hsa-mir-519a hsa-mir-520d-5p hsa-mir hsa-mir-532-3p hsa-mir-532-5p hsa-mir hsa-mir-542-3p hsa-mir hsa-mir-548c-5p hsa-mir-548d-3p hsa-mir-548d-5p hsa-mir hsa-mir-574-3p hsa-mir-576-3p hsa-mir hsa-mir-582-3p hsa-mir hsa-mir-590-5p hsa-mir hsa-mir hsa-mir hsa-mir-628-5p hsa-mir hsa-mir hsa-mir hsa-mir hsa-mir hsa-mir hsa-mir-671-3p hsa-mir hsa-mir hsa-mir-885-5p hsa-mir-886-5p hsa-mir hsa-mir-891a hsa-mir-92a hsa-mir hsa-mir hsa-mir hsa-mir hsa-mir

16 hsa-mir-99a hsa-mir-99b dme-mir hsa-let-7e# hsa-let-7f-1# hsa-let-7f-2# hsa-mir-100# hsa-mir-106b# hsa-mir hsa-mir hsa-mir-1226# hsa-mir hsa-mir hsa-mir hsa-mir hsa-mir hsa-mir-1255b hsa-mir-125b-1# hsa-mir-126# hsa-mir hsa-mir-1274a hsa-mir-1274b hsa-mir hsa-mir hsa-mir hsa-mir hsa-mir hsa-mir hsa-mir hsa-mir hsa-mir hsa-mir-130b# hsa-mir-135b# hsa-mir-148b# hsa-mir-151-3p hsa-mir-151-5p hsa-mir-15a# hsa-mir-15b# hsa-mir-16-1# hsa-mir-17# hsa-mir-181a-2# hsa-mir-183# hsa-mir-18a#

17 hsa-mir-190b hsa-mir-191# hsa-mir-192# hsa-mir-193b# hsa-mir-19a# hsa-mir-19b-1# hsa-mir hsa-mir-20a# hsa-mir-21# hsa-mir hsa-mir-22# hsa-mir-222# hsa-mir-23a# hsa-mir-24-2# hsa-mir-25# hsa-mir-26a-1# hsa-mir-26a-2# hsa-mir-26b# hsa-mir-27a# hsa-mir-27b# hsa-mir-29a# hsa-mir-29b-1# hsa-mir-29b-2# hsa-mir-30a-3p hsa-mir-30a-5p hsa-mir-30d# hsa-mir-30d hsa-mir-30e-3p hsa-mir-31# hsa-mir-320b hsa-mir-335# hsa-mir-338-5p hsa-mir-33a# hsa-mir-340# hsa-mir-34a# hsa-mir-34b hsa-mir-34b hsa-mir-374a# hsa-mir hsa-mir hsa-mir-409-3p hsa-mir-425# hsa-mir-454#

18 hsa-mir hsa-mir-505# hsa-mir-516-3p hsa-mir-520c-3p hsa-mir-520d-3p hsa-mir-545# hsa-mir hsa-mir hsa-mir hsa-mir hsa-mir hsa-mir-590-3p hsa-mir hsa-mir hsa-mir hsa-mir-625# hsa-mir-628-3p hsa-mir hsa-mir hsa-mir hsa-mir hsa-mir hsa-mir hsa-mir-744# hsa-mir hsa-mir-769-5p hsa-mir-875-5p hsa-mir-9# hsa-mir-92a-1# hsa-mir-93# hsa-mir hsa-mir hsa-mir hsa-mir-99a# hsa-mir-99b# rno-mir-29c# rno-mir-7#

19 Table S2: Primers for qrt-pcr. Sequences of the primers used for each gene are listed. 18S rrna Beta actin 5 -GTAACCCGTTGAACCCCATT-3 5 -CCATCCAATCGGTAGTAGCG-3' 5 -TCCCTGGAGAAGAGCTACGA-3' 5 -AGCACTGTGTTGGCGTACAG-3' Pri-miR-146b 5 -ATTCAGGGTTTTGGGGAGAT-3' 5 -GGGGCTTTCTGAGCTAAAGG-3' STAT3 SOCS3 BIRC3 NFKB2 RELB IKBα A20 CCL2 IL6 RELA JUNB MCL1 EGR1 KLF4 5 -ACCGGCGTCCAGTTCACTACT-3' 5 -CCGGGATCCTCTGAGAGCTGC-3' 5 -TCAAGACCTTCAGCTCCAAG-3' 5 -TGACGCTGAGCGTGAAGAAG-3' 5 -GGGAAGAGGAGAGAGAAAGAGC-3' 5 -TCCAGGATTGGAATTACACAAG-3 5 -AGAGGGAGGAGGGCCTTTAG-3' 5 -CAGGTTCTGCTTCCCAGAAT-3' 5 -AGCATCCTTGGGGAGAGC-3' 5 -AGGCAGTCACCTCCACCTC-3' 5 -ACGAGCAGATGGTCAAGGAG-3' 5 -CTTCCATGGTCAGTGCCTTT-3' 5 -CCTTGGAAGCACCATGTTTG-3' 5 -TTGTGTGGTTCGAGGCACAT-3' 5 -TGCCCCAGTCACCTGCTGTT-3' 5 -CCCACTTCTGCTTGGGGTCAGC-3' 5 -GAAAGCAGCAAAGAGGCACT-3' 5 -TTTCACCAGGCAAGTCTCCT-3' 5 -CCACGAGCTTGTAGGAAAGG-3' 5 -CTGGATGCGCTGACTGATAG-3' 5 -AAATGGAACAGCCCTTCT-3' 5 -TGTAGAGAGAGGCCACCA-3' 5 -GAGACCTTACGACGGGTT-3' 5 -TTTGATGTCCAGTTTCCG-3' 5 -AGCCCTACGAGCACCTGAC-3' 5 -AGCGGCCAGTATAGGTGATG-3' 5 -TCCCATCTTTCTCCACGTTC-3' 18

20 5 -AGTCGCTTCATGTGGGAGAG-3' BCL6 BCL3 BCL-X E-cadherin 5 -CTGCAGATGGAGCATGTTGT-3' 5 -TCTTCACGAGGAGGCTTGAT-3' 5 -CCTCTGGTGAACCTGCCTAC-3' 5 -TACCCTGCACCACAGCAATA-3' 5 -GGTATTGGTGAGTCGGATCG-3' 5 -TGCTGCATTGTTCCCATAGA-3' 5 -CCTGGGACTCCACCTACAGA-3' 5 -TGTGAGCAATTCTGCTTGGA-3' Desmoplakin 5 -GGCACCAGCAGGATGTACT-3' 5 -ATCAAGCAGTCGGAGCAGTT-3' Vimentin 5 -TCAGAGAGAGGAAGCCGAAA-3' 5 -ATTCCACTTTGCGTTCAAGG-3' Cadherin CAACGGACTATGAAACACAGGA-3' 5 -GAAAGGGCCATTGCTGATAA-3' Slug 5 -TCGGACCCACACATTACCTT-3' 5 -TGACCTGTCTGCAAATGCTC-3' 19